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1.
Bioorg Med Chem Lett ; 24(13): 2885-91, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24835984

RESUMO

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4-TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.


Assuntos
Descoberta de Drogas , Proteínas Plasmáticas de Ligação ao Retinol/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Ligantes , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Ratos , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Vitamina A/sangue
2.
Proc Natl Acad Sci U S A ; 108(18): 7379-84, 2011 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-21502526

RESUMO

Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.


Assuntos
Amidoidrolases/antagonistas & inibidores , Benzimidazóis/isolamento & purificação , Benzimidazóis/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Animais , Benzimidazóis/farmacocinética , Cumarínicos , Cristalização , Inibidores Enzimáticos/farmacocinética , Escherichia coli , Humanos , Estrutura Molecular , Ratos , Espectrofotometria Ultravioleta , Espectrometria de Massas em Tandem , Ureia/metabolismo
3.
J Clin Invest ; 133(12)2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37317970

RESUMO

While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.


Assuntos
Imunidade Adaptativa , Neutrófilos , Humanos , Citocinas , Terapia de Imunossupressão , Imunoterapia
4.
MAbs ; 12(1): 1710047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31905038

RESUMO

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Aumento de Peso/efeitos dos fármacos , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , Conformação Proteica
5.
Bioorg Med Chem ; 16(19): 8922-31, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18789704

RESUMO

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the NADPH dependent interconversion of inactive cortisone to active cortisol. Excess 11beta-HSD1 or cortisol leads to insulin resistance and metabolic syndrome in animal models and in humans. Inhibiting 11beta-HSD1 activity signifies a promising therapeutic strategy in the treatment of Type 2 diabetes and related diseases. Herein, we report two highly potent and selective small molecule inhibitors of human 11beta-HSD1. While compound 1, a sulfonamide, functions as a simple substrate competitive inhibitor, compound 2, a triazole, shows the kinetic profile of a mixed inhibitor. Co-crystal structures reveal that both compounds occupy the 11beta-HSD1 catalytic site, but present distinct molecular interactions with the protein. Strikingly, compound 2 interacts much closer to the cofactor NADP+ and likely modifies its binding. Together, the structural and kinetic analyses demonstrate two distinctive molecular inhibition mechanisms, providing valuable information for future inhibitor design.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Doenças Metabólicas/patologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Sequência de Aminoácidos , Sítios de Ligação , Bioensaio , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Cinética , Doenças Metabólicas/enzimologia , Dados de Sequência Molecular , NADP/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Triazóis/química , Triazóis/farmacologia
6.
Structure ; 21(5): 798-809, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23602659

RESUMO

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.


Assuntos
Lisofosfolipídeos/química , Esfingosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Humanos , Cinética , Lisofosfolipídeos/metabolismo , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Esfingosina/química , Esfingosina/metabolismo , Especificidade por Substrato
7.
J Med Chem ; 55(8): 3837-51, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22458568

RESUMO

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Guanina/análogos & derivados , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacologia , Organofosfonatos/síntese química , Capuzes de RNA/metabolismo , Animais , Cristalografia por Raios X , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/química , Guanina/síntese química , Guanina/farmacologia , Guanosina Monofosfato/síntese química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Organofosfonatos/farmacologia , Ácidos Fosforosos , Biossíntese de Proteínas/efeitos dos fármacos , Capuzes de RNA/química , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Relação Estrutura-Atividade
8.
J Biol Chem ; 284(12): 7673-80, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19147488

RESUMO

Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (K(i) = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4(-/-) mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes.


Assuntos
Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Piperidinas/química , Proteínas Plasmáticas de Ligação ao Retinol , Vitamina A/sangue , Animais , Cristalografia por Raios X , Diabetes Mellitus/sangue , Diabetes Mellitus/tratamento farmacológico , Gorduras na Dieta/efeitos adversos , Humanos , Insulina/metabolismo , Resistência à Insulina , Ligantes , Camundongos , Camundongos Knockout , Piperidinas/farmacologia , Estrutura Terciária de Proteína , Proteínas Plasmáticas de Ligação ao Retinol/agonistas , Proteínas Plasmáticas de Ligação ao Retinol/química , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo
9.
J Biol Chem ; 283(14): 9168-76, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18263587

RESUMO

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma by TZDs improves insulin sensitivity; however, this is accompanied by the induction of several undesirable side effects. We have identified a novel synthetic PPARgamma ligand, T2384, to explore the biological activities associated with occupying different regions of the receptor ligand-binding pocket. X-ray crystallography studies revealed that T2384 can adopt two distinct binding modes, which we have termed "U" and "S", interacting with the ligand-binding pocket of PPARgamma primarily via hydrophobic contacts that are distinct from full agonists. The different binding modes occupied by T2384 induced distinct patterns of coregulatory protein interaction with PPARgamma in vitro and displayed unique receptor function in cell-based activity assays. We speculate that these unique biochemical and cellular activities may be responsible for the novel in vivo profile observed in animals treated systemically with T2384. When administered to diabetic KKAy mice, T2384 rapidly improved insulin sensitivity in the absence of weight gain, hemodilution, and anemia characteristics of treatment with rosiglitazone (a TZD). Moreover, upon coadministration with rosiglitazone, T2384 was able to antagonize the side effects induced by rosiglitazone treatment alone while retaining robust effects on glucose disposal. These results are consistent with the hypothesis that interactions between ligands and specific regions of the receptor ligand-binding pocket might selectively trigger a subset of receptor-mediated biological responses leading to the improvement of insulin sensitivity, without eliciting less desirable responses associated with full activation of the receptor. We suggest that T2384 may represent a prototype for a novel class of PPARgamma ligand and, furthermore, that molecules sharing some of these properties would be useful for treatment of type 2 diabetes.


Assuntos
Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Benzotiazóis/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , PPAR gama/agonistas , Sulfonamidas/farmacologia , Adipócitos/patologia , Animais , Benzotiazóis/química , Benzotiazóis/uso terapêutico , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Ligantes , Camundongos , Modelos Moleculares , PPAR gama/química , PPAR gama/metabolismo , Rosiglitazona , Sulfonamidas/química , Sulfonamidas/uso terapêutico , Tiazolidinedionas/química , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Aumento de Peso/efeitos dos fármacos
10.
Chem Biol Drug Des ; 71(1): 36-44, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18069989

RESUMO

11Beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid action and inhibition of this enzyme is a viable therapeutic strategy for the treatment of type 2 diabetes and the metabolic syndrome. Here, we report a potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor with a binding mode elucidated from the co-crystal structure with the human 11beta-hydroxysteroid dehydrogenase type 1. The inhibitor is bound to the steroid-binding pocket making contacts with the catalytic center and the solvent channel. The inhibitor binding is facilitated by two direct hydrogen bond interactions involving Tyrosine183 of the catalytic motif Tyr-X-X-X-Lys and Alanine172. In addition, the inhibitor makes many hydrophobic interactions with both the enzyme and the co-factor nicotinamide adenine dinucleotide phosphate (reduced). In lean C57BL/6 mice, the compound inhibited both the in vivo and ex vivo 11beta-hydroxysteroid dehydrogenase type 1 activities in a dose-dependent manner. The inhibitory effects correlate with the plasma compound concentrations, suggesting that there is a clear pharmacokinetic and pharmacodynamic relationship. Moreover, at the same doses used in the pharmacokinetic/pharmacodynamic studies, the inhibitor did not cause the activation of the hypothalamic-pituitary-adrenal axis in an acute mouse model, suggesting that this compound exhibits biological effects with minimal risk of activating the hypothalamic-pituitary-adrenal axis.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Tiazóis/administração & dosagem , Tiazóis/farmacocinética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/enzimologia , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína
11.
Nature ; 423(6939): 555-60, 2003 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-12774125

RESUMO

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Proteínas Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas Oncogênicas/metabolismo , Especificidade de Órgãos , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Células Tumorais Cultivadas
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